Dynamics of extended-spectrum cephalosporin resistance genes in Escherichia coli from Europe and North America

Extended-spectrum cephalosporins (ESCs) are critically important antimicrobial agents for human and veterinary medicine. ESC resistance (ESC-R) genes have spread worldwide through plasmids and clonal expansion, yet the distribution and dynamics of ESC-R genes in different ecological compartments are poorly understood. Here we use whole genome sequence data of Enterobacterales isolates of human and animal origin from Europe and North America and identify contrasting temporal dynamics. AmpC β-lactamases were initially more dominant in North America in humans and farm animals, only later emerging in Europe. In contrast, specific extended-spectrum β-lactamases (ESBLs) were initially common in animals from Europe and later emerged in North America. This study identifies differences in the relative importance of plasmids and clonal expansion across different compartments for the spread of different ESC-R genes. Understanding the mechanisms of transmission will be critical in the design of interventions to reduce the spread of antimicrobial resistance.

The CAN-ICU Study was conducted from September 2005 to June 2006 at 19 medical centres with active ICUs from across Canada. Centres submitted clinically relevant isolates (one pathogen per cultured site per patient) from patients in medical, surgical, and pediatric ICUs. Participating centres were asked to submit 300 consecutive clinically significant bacterial pathogens isolated from blood, urine, wound, and respiratory tract specimens. Isolates were deemed clinically significant according to each submitting laboratory's own standard protocol.
Genomic DNA for whole genome sequencing (WGS) was extracted from cultures grown overnight at 37°C in Luria Broth, using the Epicentre MasterPure™ Complete kits (Mandel Scientific, Guelph, ON, Canada). Isolates were then submitted to the Genomics Core Facility at the Public Health Agency's National Microbiology Laboratory for library preparation and sequencing. Paired-end libraries were constructed using the Nextera XT Library Prep Kit (Illumina, San Diego, CA) and 150-bp paired-end indexed reads were generated on the Illumina MiSeq platform. Raw sequences were provided by the Genomics Core Facility for further analysis.

Animal and food isolates from France
Non-human isolates came either from the Resapath network that collects bacteria from sick animals (all included isolates from cats, dogs, horses and the majority of isolates from cattle), from specific surveillance studies at the slaughterhouse (n=32 E. coli from cattle) and from one specific study on chicken meat. Bacteria from the Resapath were isolated in peripheral veterinary laboratories on non-selective media. Bacteria from the two specific studies were isolates on selective ChromID ESBL medium (Biomérieux, Marcy l'Etoile, France). DNA from all isolates was extracted using the NucleoSpin Microbial DNA mini kit (Macherey-Nagel, Hoerdt, France). WGS was outsourced to the University of Clermont-Ferrand and DNA were processed as described above for French human isolates.

Animal isolates from Germany
The 199 isolates from Germany were obtained from the strain collection of the national antimicrobial resistance monitoring program of animal pathogenic bacteria in Germany, GERM-Vet, conducted by the Federal Office of Consumer Protection and Food Safety (BVL), Berlin, Germany. Veterinary diagnostic laboratories from all over Germany provide preidentified bacteria from acutely diseased animals, which have not been treated with antimicrobial agents in the six weeks prior to sampling, according to a defined sampling plan to this program. When the bacteria arrive at BVL, they are inoculated on non-selective media (i.e. sheep blood agar plates (Oxoid, Wesel, Germany)) for purity checks and are confirmed for their species assignment by MALDI-TOF mass spectrometry (Bruker, Bremen, Germany). Thereafter, the bacteria were subjected to antimicrobial susceptibility testing (AST) by broth microdilution according to CLSI standards using commercial microtiter plates (MSC diagnostics Swalmen, The Netherlands). The panel of antimicrobial agents tested included βlactams (penicillin, ampicillin, amoxicillin-clavulanic acid, ceftiofur, cefquinome, cephalothin, cefotaxime, cefoperazone), macrolides (tilmicosin, tulathromycin), (fluoro)quinolones (nalidixic acid, ciprofloxacin, enrofloxacin, marbofloxacin), tetracyclines (tetracycline, doxycycline), aminoglycosides (gentamicin, neomycin, streptomycin), a phenicol (florfenicol), a pleuromutilin (tiamulin), a polypeptide (colistin) and the combination of folate pathway antagonists trimethoprim-sulfamethoxazole. Escherichia coli ATCC25922 served as a quality control strain in AST.
DNA of the German isolates was prepared using the MasterPure DNA Purification Kit (Epicentre, Madison, WI, USA) according to the manufacturer's recommendations. DNA libraries were prepared using the Nextera XT Library Preparation Kit (Illumina, San Diego, CA, USA) as recommended by the manufacturer. Sequencing was performed on the Illumina MiSeq (Illumina) platform using the Miseq Reagent Kit v3 and the Nextera XT index kit2 v2 with an approximately 100-fold coverage.

Section 2: Long-read data and hybrid assembly
Long-read sequencing of 20 isolates was performed using a MinION (Oxford Nanopore Technologies, Oxford, United Kingdom). Base-calling of the fast5 files and demultiplexing of the Nanopore reads were performed using Guppy Basecaller v4.5.4 and Guppy Barcoder v4.5.4 (Oxford Nanopore Technologies), respectively. Adapters from long reads were removed with Porechop v0.2.4 5 . Filtlong v0.2.0 (https://github.com/rrwick/Filtlong) was used to remove reads <2Kpb in length and low quality reads; read files with a very large size were reduced to a 500 Mbp subset. Hybrid assembly using short and long reads for each isolate was obtained with Unicycler v0.4.8 6 using the "bold" mode option to get complete assembly. Hybrid assemblies were polished using the Illumina reads with Pilon v1.22 7 up to five or ten cycles of polishing. The mapping of the ESC-R genes to chromosome or plasmids was done using Abricate v0.9.8 (https://github.com/tseemann/abricate) and the Resfinder 8 database.
Section 3: Plasmidome analysis of plasmids linked with ESC-R genes from short-read data The predicted plasmid contigs (recovered from short-read data) harboring the main ESC-R genes were annotated using Prokka v1.13 9 . With Roary v3.12.0 10 , the core genes were identified with 90% sequence similarity and presence in 95% of isolates. The Roary matrix of gene presence absence was used to generate a gene content (plasmidome) network through the PANINI 11 web tool. The plasmidome network was annotated with the metadata (ESC-R gene, plasmid Inc. type, plasmid subtypes, country, source and year) and then visualized. In this analysis, all 313 typeable ESC-R plasmid contigs were included (136 blaCTX-M-1, 23 blaCTX-M-15, 88 blaCMY-2, 40 blaCTX-M-14 and 26 blaSHV-12) plus 79 non-typeable plasmids. The latter represents a subset of the 823 non-typeable plasmids identified, and were selected using the following criteria: they were predicted as plasmids by both MOB-suite (which reconstructs plasmids) and RFPlasmid, and so should be longer sequences and thus contain more information than those non-typeable plasmids predicted by RFPlasmid alone. The 79 nontypeable plasmids were added in the plasmidome analysis to investigate how they relate to the typeable plasmids. Section 4: Pangenome analysis of 1,818 E. coli genomes Similar to the analysis described in the above section, the gene presence absence matrix obtained from 1,818 E. coli genomes using Roary was used to generate the gene context network, which was annotated with the metadata such as phylogroup, main ST/CC, ESC-R, country, source and year.                          105 (75) *Plasmid and chromosome origin by MOB-suite and RFPlasmid consist of: i) chromosome category as inferred by both MOB-suite and RFPlasmid, ii) plasmid category includes typeable plasmids (presence of plasmid replicon genes) and non-typeable plasmids (absence of plasmid replicon genes); the typeable plasmid (ESC-R plasmids) category included plasmid prediction by both MOB-suite and RFPlasmid; the non-typeable plasmid category included also those classified as plasmids by RFPlasmid but as chromosome by MOB-suite. a blaCMY-2: in three genomes the ESC-R gene location was predicted as chromosome by RFPlasmid but as plasmid by MOB-suite. Inspection of these contigs identified replicon sequences, therefore they were included within the plasmid category. b blaCTX-M-14: one chromosome inference by RFPlasmid but predicted as plasmid by MOBsuite. This contig also carried a replicon sequence, therefore it was included within the plasmid category.   Table S11).   *Q-FQ (mutations); resistance to quinolone-fluoroquinolone due to mutations in gyrA, parC and parE. **Quinolone-FQ; resistance to quinolone-fluoroquinolone due to acquired AMR genes.  gyrA S83A     **Cases where two distinct replicons were found in the same plasmid (i.e., ColRNAI+IncI1, IncF+IncI1, IncI1+rep, rep+IncH). The main plasmid types are highlighted in the table and the dash "-" indicate of the absence of the data.